The first objective of this research is to study the transfer of lipids, amphiphiles and membrane proteins across and between phospholipid bilayers. In vivo, these processes occur during membrane turnover, and in the metabolism of phospholipids, fats, steroids, lipid-soluble vitamins, porphyrins, certain drugs and environmental toxins. However, the mechanism is not clear. We plan to study the kinetics and equilibrium distribution of these transfers in liposomes, with primary attention given to the forces that may catalyze flip-flop of phospholipid, cholesterol and membrane peptides. Some possible effectors: membrane proteins, transbilayer mass imbalances, divalent ions and minor lipid components. Flip-flop and intervesicle transfer can be measured by chemical titration and/or separation of vesicle populations by sedimentation or ion exchange. Cytochrome b5 is an intrinsic membrane protein that, in vivo, is probably synthesized in the aqueous phase, then binds spontaneously to intracellular membranes. In vitro studies show that this protein can exchange between liposomes, and perhaps between microsomes and liposomes. To understand the subcellular distribution of this protein, the binding of cyt b5 to liposomes of varying composition will be studied. Also, the effect of this protein on lipid flip-flop and transbilayer compositional asymmetry will be determined. The second objective of this research is to begin the purification and characterization of one of the key membrane proteins in phospholipid biosynthesis, diacyl glycerol phosphoethanolamine transferase (EC 2.7.8.1). If successful, this enzyme will be reconstituted in liposomes. The mechanism of the enzyme and the physical properties of the reconstituted system will be studied in detail.